Diagnostic aid



United States Patent 3,260,648 DIAGNUSTIC AID Charles Fox, Fair Lawn, N.J., assignor to Warner-Lambert Pharmaceutical Company, Morris Plains, N.J., a corporation of Delaware No Drawing. Filed Aug. 16, 1963, Ser. No. 302,702 8 (llaims. (Cl. 167-84) This invention relates to a new and improved diagnostic composition for use in the determination of total cholesterol levels in human blood plasma. More particularly, the present invention relates to a novel lyophilized human blood plasma composition containing a known and standardized amount of total cholesterol which is solubilized in said composition for certain solubilizing agents. Specifically, the present invention relates to certain solubilizing agents which do not interfere to any material degree with the usual test procedures performed with a novel diagnostic composition containing total cholesterol when the diagnostic composition is reconstituted to solution form.

Cholesterol, either in the free form or as an ester, is an essential constituent of all animal and plant cells, and is often accompanied by its derivatives, dihydrocholesterol and 7-dihydrocholesterol. Blood cholesterol both esterified and free is one of the major blood lipids and is present together with total fatty acids, triglycerides, and phosphatides. Cholesterol in the blood is distributed almost equally between the plasma and corpuscles, with higher concentrations in the leukocytes. Human blood plasma normally contains 100 to 320 mg. of total cholesterol, with a mean of 160 mg. per 100 cc. of plasma which is usually so distributed that about one-fourth is free cholesterol and the remaining three-fourths as the cholesterol ester of fatty acids. Since the sum of the free cholesterol and the cholesterol esters in the serum is usually referred to as total cholesterol, for convenience, this term will be so applied hereinafter.

*Every human being maintains a constant total serum cholesterol level and large deviations from this level do not ordinarily occur. However, research directed to the effects of abnormally high total cholesterol blood levels has made it increasingly apparent that the total cholesterol blood level should be controlled and should be maintained within the limits considered normal for the particular individual. The total cholesterol level in the blood plasma tends to be high, for example, in hyperglycemia and in uncontrolled diabetes. Cholesterol has also been found to be the principal lipid in the intirna of the arterial wall in atherosclerosis. Plasma cholesterol levels are also high in icterus, naphritis, alcoholism, anesthesia, pregnancy, syphilis, and following excision of the adrenals. Total serum cholesterol is also elevated in diseases of the liver associated with obstruction of bile flow, in familial hypercholesterolemia, in hypothyroidism and in the nephrotic syndrome. Low blood cholesterol is found in severe liver disease, wasting illness, malnutrition, and hyperthyroidism. Accordingly, the accurate determination of serum cholesterol levels is important for modern diagnostic procedures.

In clinical laboratories the use of lyophilized blood plasma as a diagnostic aid is well known. An example of one such diagnostic aid which is employed as a blood serum standard in blood chemistry analysis is one consisting of pooled human sera containing standardized amounts of the protein solids of human blood plasma including bilirubin, uric acid, thyroglobulin, glycerin, urea, dextrose, cereatinine along with minor amounts of various sodium and potassium compounds. These compositions are referred to hereinafter as lyophilized human blood plasma.

Patented July 12, 1966 ice While cholesterol may be found in diagnostic aids comprising lyophilized human blood plasma, the quantity of total cholesterol varies with the level of cholesterol in the pooled blood. Accordingly, the lyophilized blood plasma cannot serve as a standardized diagnostic aid in clinical test procedures for determining serum cholesterol blood levels. Therefore, it is desirable to provide a pooled human blood plasma standardized for total cholesterol at a known level so that the cholesterol level in a blood serum test sample may be readily determined by comparison with a standardized sample. The difficulty which resides in simply adding cholesterol to such diagnostic aids is that the cholesterol does not readily dissolve on reconstitution of the lyophilized blood plasma with Water. When cholesterol does not readily dissolve in the reconstitution of blood plasma, it materially interferes with analytical test procedures which are performed with the aid of the reconstituted lyophilized blood plasma other than the total cholesterol level.

A diagnostic composition and method for using same, to be of greatest value must be rapid, accurate, and simple enough to be learned with case. All of these advantages have not been achieved heretofor in a diagnostic composition containing a standard cholesterol level primarily because total cholesterol is difficult to solubilize in reconstituted lyophilized blood plasma compositions. Although many solubilizing agents are known to be useful in solubilizing total cholesterol in various fluids including human blood, it has been observed that these agents materially interfere with accuracy of the analytical tests ordinarily performed in the preferred blood chemistry analytical procedures. A need is therefore established for a diagnostic composition having a standardized total cholesterol level which composition may be readily dissolved on reconstitution and will not materially interfere with the usual procedures for determining total cholesterol blood levels.

It is an important object of this invention therefore to provide a lyophilized blood plasma for use as a diagnostic aid containing predetermined and known amounts of total cholesterol in readily solubilized form.

It is another object of this invention to provide certain cholesterol solubilizing agents which are compatible with the total cholesterol in an aqueous body fluid and which will not adversely interfere with analytical tests performed on said body fluid.

It is a further object of this invention to provide an optical method for the determination of the total cholesterol present in a sample of human blood by an optical comparison method employing a reconstituted diagnostic aid containing a predetermined and standardized amount of solubilized total cholesterol.

These and other objects will become apparent from the following detailed description of the present invention which generally provides an improved, standardized diagnostic aid and to methods for using same, which when reconstituted in an aqueous medium and with the desired total cholesterol level has an optical density of less than 0.450 at about 620 Inn. This degree of clarity is obtained when total cholesterol present in the lyophilized human blood plasma is solubilized by the addition of an agent of the following general formula:

RO (CH CH O H wherein R is a straight or branched chain alkyl group of from C to about C a lower alkyl phenoxy group or an acyl group containing 12 to 22 carbon atoms; and n is an integer from 10 to about 20. An embodiment of the present invention provides a lyophilized human blood plasma for use as a diagnostic aid containing a solubilizing agent for total cholesterol as defined above wherein the solubilizing agent is present in a ratio from about a) 7.5 to about parts by weight of solubilizing agent for each part by weight of total cholesterol.

In the practice of this invention, pooled human blood plasma is processed by reducing the amount of basic constituents normally present to a fixed low level. Thereafter, the same constituents, in purified form, are returned to the plasma and fixed to a predetermined level. When this product containing fixed levels of the various constituents is combined with certain additives, a diagnostic aid in the form of a material suitable for use as a primary standard in blood chemistry analysis is thereupon obtained. After the diagnostic aid is prepared, total cholesterol may be added thereto along with the solubilizing agents of the present invention. The solubilizing agent may be blended directly with a predetermined quantity of hot distilled water and may then be added in a quantity equal to the weight of the total cholesterol blend. The hot water which is added is desirably maintained at a temperature of about 120 to about 200 F. to facilitate solubilization of total cholesterol. The amount of solubilizin-g agent added to the total cholesterol is within the weight ratio of about 7.5 :1 to about 15:1, as indicated above, and is preferably within the range of from about 8:1 to about 10:1. This cholesterol solution may then be added directly to the processed human blood plasma, and the solution is thereafter lyophilized.

When the solubilized cholesterol is added directly as a solution to processed human blood plasma, the added solution conveniently contains about 2% to about 5% by weight of total cholesterol solution. The amount of total cholesterol added to the processed human blood plasma, however, will necessarily depend upon the total cholesterol level desired in the lyophilized product. As an alternate procedure, the solubilized cholesterol may be added to the reconstituted blood plasma prior to testing. The standardized reconstituted blood plasma reagent may contain from 75 to 500 mg./100 cc. of total cholesterol and preferably from 100 to 325 mg./100 cc. of total cholesterol. The ratio of the cholesterol to the cholesterol acetate in the cholesterol content of the test reagent is not critical and usually varies from about 0.1 to about 10.0. A desirable ratio, however, is from 0.5 to about 1.5.

The following examples are included in order further to illustrate the invention. All parts are given by weight unless otherwise specified.

Example 1 The following ingredients are weighed into a 250 ml. beaker and uniformly blended at 212 F.:

Ingredient: Parts by weight, gms.

When the above ingredients are thoroughly blended in a mixer, the mixture is cooled to 180 F. Twentyfive milliliters of distilled water at a temperature of 180 F. are added to the blend which is then allowed to gradually cool to room temperature with mild stirring. An additional amount of distilled water is added, if necessary, to form a solution which measures 100.0 milliliters exactly. Five milliliters of this blended solution is added to a milliliter vial of human blood plasma and 1yophilized. When reconstituted, the lyophilized blood plasma is found to have a total cholesterol level of 310 milligrams/100 milliliters of reconstituted plasma.

For determining the total cholesterol in an unknown sample, pipet 0.5 milliliter samples of the above prepared standard reconstituted blood serum with a similar volume of an unknown sample taken from a patient and place these samples into labeled 50 milliliter glass-stoppered 4- eentr-ifuge tubes. Five milliliters of an alcoholic potassium hydroxide solution (6 milliliters of a 33% aqueous potassium hydroxide solution in 94 milliliters absolute alcohol) are added to each sample. The tubes are stoppered, shaken well, and incubated at 3740 C. for 55 minutes whereupon they are cooled to room temperature. At room temperature, 10 milliliters of petroleum ether are added to each tube and mixed for about one minute with 5 milliliters of Water. The samples are then placed in a centrifuge at slow speed for 5 minutes or until the emulsion breaks and two clear layers are formed. A four milliliter aliquot of the less dense top layer containing the petroleum ether is removed from each tube and transferred to small dry flasks and the petroleum ether is evaporated by warming the flasks and gently blowing a stream of air into them. When dry, the samples are cooled to room temperature.

At room temperature 6 milliliters of modified Liebermann-Burchard reagent are added to the flask containing the residue at timed intervals. The modified Liebermann- Burchard reagent is prepared from 20 volumes of acetic anhydride, 1 volume of concentrated sulfuric acid and 10 volumes of glacial acetic acid. The samples are read for optical density of a Beckman DU spectrophotometer against a clear water blank at 620 m exactly 30 minutes after adding the Liebermann-Burchard reagent. The optical density of the standard having 310 milligrams of total cholesterol/ milliliters of reconstituted plasma is found to have an optical density at 620mg of 0.380. The optical density of the unknown is found to be 0.163 at 620 III/L. The total cholesterol level in the unknown sample when determined by direct ratio of the optical densities to the total cholesterol levels is found to be 200 milligrams/100 milliliters of plasma.

Example 2 The following ingredients are weighed into a 350 ml. beaker and uniformly blended at 212 F.:

Parts by Ingredient: weight, gms. Cholesterol U.S.P. 0,125 Cholesterol acetate 0.375

Oleyl alcohol condensate (average 10 mol ethylene oxide/mol) 10.0 Oleyl alcohol condensate (average 20 mol ethylene oxide/mol) 10.0 Polyoxyethylene polyoxypropylene condensate 5.0

The above ingredients are processed as in Example 1. The lyophilized mass obtained reconstitutes to a clear solution within 30 minutes with no foaming or undue bubbling. The total cholesterol level is tested and found to be 300 milligrams/100 milliliters of the reconstituted plasma. The reconstituted solution is tested at 72 F. for optical density on a Beckman DU spectrophotometer as described in Example 1 and is found to have an optical density of 0.400 with a clear water blank at 620 m l.

Example 3 The following ingredients are weighed into a 250 ml.

The above ingredients are processed as in Example 1. The lyophilized mass reconstitutes to a clear solution within 30 minutes with no foaming or undue bubbling. The total cholesterol level of the reconstituted plasma is tested and found to be 300 milligrams/100 milliliters of reconstituted plasma. The reconstituted solution is tested as previously in Example 1 at 72 F. for optical density on a Beckman DU spectrophotometer and is found to have an optical density with a clear water blank at 620 m of 0.380.

Example 4 The following ingredients are weighed into a 250 ml. beaker and uniformly blended at 212 F.:

The above ingredients are processed as in Example 1. The lyophilized mass reconstituted to clear solution within 30 minutes with no foaming or undue bubbling. The total cholesterol level is tested and found to be 300 milligrams/ 100 milliliters of reconstituted plasma. The reconstituted solution is tested as previously in Example 1 at 72 F. for optical density on a Beckman DU spectrophotometer and is found to have an optical density with a clear water blank at 620 m of 0.380.

Although the method for determining solubilized total cholesterol in accordance with this invention has been specifically disclosed for use in diagnostic aid materials, it may also be applied in the case of medicinal bases, cosmetic bases, lotions, creams, reconstituted powders and the like without departing from the practice of this invention. With slight modifications in the procedure disclosed herein, the practice of the present invention may also be applicable to the solubilization of steroids in addition to total cholesterol. Thus, other steroids may be included such as, for example, those characterized by the fact that they yield methylcyclopentenophenanthrene (Diels hydrocarbon) on dehydrogenation with selenium. To this group belongs the sterols such as the zoosterols and phytosterols; the bile acids; the cardiac aglycones; the sex hormones, the adrenal steroids and the steroid sapagenins. In addition to cholesterol, examples of the zoosterols include materials such as coprosterol, 7-dehydrocholesterol and the like. The phytosterols include materials such as stigmasterol, B-sitosterol, ergosterol, calciferol and the like. The bile acids include materials such as cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid and the like. The cardiac aglycones include materials such as digitoxiginin, strophanthidin, scillaren-A and the like. The sex hormones include materials such as testosterone, estradiol, stilbestrol, hexestrol, progesterone and the like. The adrenal steroids include materials such as corticosterone, deoxycorticosterone, cortisone, aldosterone, and the like. The steroid sapagenins include materials such as dios-genin, tigogenin, digitonin and the like.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of this invention.

What is claimed is:

1. A method for the determination of the total cholesterol level in blood plasma comprising,

(A) reconstituting a lyophilized human blood plasma containing weighed-in total cholesterol in known and standardized amount in combination with a solubilizing agent of the following general formula:

R0 (CH CH O) H wherein R is selected from the group consisting of: (a) a straight chain alkyl group of from about C to about C (b) a branched chain alkyl group of from about C12 to about C18,

(c) a lower alkyl phenoxy group, and (d) an acyl group of from 12 to 22 carbon atoms; and n is an integer from about 10 to about 20;

(B) adding the reconstituted human blood plasma to alcoholic potassium hydroxide to liberate the solubilized cholesterol from lipoprotein complexes contained therein;

(C) extracting the liberated cholesterol with a petroleum ether;

(D) measuring the cholesterol level in the petroleum ether by reading the optical density of the sample against a blank after adding Liebermann-Burchard reagent; and

(E) calculating the cholesterol level of an unknown sample by comparison of the optical density of the known cholesterol sample with the optical density of the unknown cholesterol sample.

2. A diagnostic aid comprising, lyophilized human blood plasma; cholesterol and cholesterol esters of fatty acids in a known and standardized amount and a solubilizing agent of the following general formula:

RO (CH CH O H wherein R is selected from the group consisting of (a) a straight chain alkyl group of from about C to about C (b) a branched chain alkyl group of from about C to about C (0) a lower alkyl phenoxy, and

(d) an acyl group of from 12 to 22 carbon atoms; and n is an integer from about 10 to about 20, said diagnostic aid having an optical density at 620 m of less than 0.450 when reconstituted to the original volume and solution.

3. A lyophilized human blood plasma as in claim 2 having a weight ratio of from about 1.5:1 to about 15:1 parts of solubilizing agent to the combined cholesterol and cholesterol acetate which when reconstituted to the original volume and solution has an optical density at 620 mp of less than 0.450.

4. A lyophilized human blood plasma as in claim 2 wherein the solubilizing agent is of the general formula:

RO (CH CH O H wherein R is selected from the group consisting of (a) a straight chain alkyl group of from about C to about C (b) a branched chain alkyl group .of from about C to about C13,

(c) a lower alkyl phenoxy, and

(d) an acyl group of from 12 to 22 carbon atoms; and n is an integer from about 10 to about 20.

5. A diagnostic aid for the determination of total cholesterol levels in blood plasma which comprises, lyophilized human blood plasma, a predetermined quantity of total cholesterol and a solubilizing agent comprising oleyl alcohol condensate having an average of about 10 moles to about 20 moles of ethylene oxide per mole of condensate.

6. A diagnostic aid for the determination of total cholesterol levels in blood plasma which comprises, lyophilized human blood plasma, a predetermined quantity of total cholesterol and a solubilizing agent comprising oleyl alcohol condensate having an average of about 10 moles of ethylene oxide per mole of said condensate and polyoxyethylene-polyoxypropylene condensate.

7. A diagnostic aid for the determination of total cholesterol levels in blood plasma which comprises, a lyophilized human blood plasma, a predetermined quantity of total cholesterol and a solubilizing agent comprising nonyl-phenoxy polyoxyethylene ether having an average of about 10 moles of ethylene oxide per mole of said nonyl-phenoxy polyoxyethylene ether, stearyl polyoxyethylene ether having an average of about 20 moles of ethylene oxide per mole of said stearyl polyoxyethylene ether, and polyethylene glycol 400 mono-oleate.

8. A diagnostic aid for the determination of total cholesterol levels in blood plasma which comprises, lyophilized human blood plasma, a predetermined quantity of total cholesterol and a solubilizing agent comprising nonyl-phenoxy polyoxyethylene ether having an average of about 10 moles of ethylene oxide per mole of said nonyl-phenoxy poly-oxyethylene ether and oleyl polyoxyethylene ether having an average of about 20 moles of ethylene oxide per mole of said oleyl polyoxyethylene ether.

No references cited,

LEWIS GOTTS, Primary Examiner.

SHEP K. ROSE, Assistant Examiner. 

1. A METHOD FOR THE DETERMINATION OF THE TOTAL CHOLESTERROL LEVEL IN BLOOD PLASMA COMPRISING, (A) RECONSTITUTING A LYOPHILIZED HUMAN BLOOD PLASMA CONTAINING WEIGHED-IN TOTAL CHOLESTEROL IN KNOW AND STANDARDIZED AMOUNT IN COMBINATION WITH A SOLUBILIZING AGENT OF THE FOLLOWING GENERAL FORMULA: 